Effective diagnosis and surveillance of complex multi-factorial disorders such as cancer can be improved by screening of easily accessible biomarkers. Highly stable cell free Circulating Nucleic Acids (CNA) present as both RNA and DNA species have been discovered in the blood and plasma of humans. Correlations between tumor-associated genomic/epigenetic/transcriptional changes and alterations in CNA levels are strong predictors of the utility of this biomarker class as promising clinical indicators. Towards this goal microRNAs (miRNAs) representing a class of naturally occurring small non-coding RNAs of 19-25 nt in length have emerged as an important set of markers that can associate their specific expression profiles with cancer development. In this study we investigate some of the pre-analytic considerations for isolating plasma fractions for the study of miRNA biomarkers. We find that measurement of circulating miRNA levels are frequently confounded by varying levels of cellular miRNAs of different hematopoietic origins. In order to assess the relative proportions of this cell-derived class, we have fractionated whole blood into plasma and its ensuing sub-fractions. Cellular miRNA signatures in cohorts of normal individuals are catalogued and the abundance and gender specific expression of bona fide circulating markers explored after calibrating the signal for this interfering class. A map of differentially expressed profiles is presented and the intrinsic variability of circulating miRNA species investigated in subsets of healthy males and females.
A considerable proportion of the animal genome representing both DNA and coding/non-coding RNAs can be detected in circulation. Identified first in 1948 and thought to originate as products of apoptosis or active release from cells, extracellular circulating DNA fragments ranging in size between 500 bp to greater than 30 Kb have been characterized both in normal and diseased individuals (see, Fleischhacker and Schmidt (2007) Biochim Biophys Acta 1775: 181-232 and van der Vaart and Pretorius P J (2007) Clin Chem 53: 2215). Although the physiological functions of these circulating species are unclear, the presence of tumor associated genetic alterations in these molecules combined with inherent molecular stability makes them attractive substrates for disease detection, tracking and prediction. Of the various classes of circulating nucleic acids—miRNAs representing approximately 1-2% of the known genes in eukaryotes (see John et al. (2004) PLoS Biol 2: e363) and characterized by highly conserved small non-coding RNAs of 19-25 nt in length are particularly attractive candidates. Approximately 940 mature miRNAs have been characterized to date in humans (see, Griffiths-Jones (2004) Nucleic Acids Res 32: D109-111, Griffiths-Jones et al. (2006) Nucleic Acids Res 34: D140-144 and Griffiths-Jones et al. (2008) Nucleic Acids Res 36: D154-158) and it is believed that approximately 30% of all annotated human genes may potentially be targeted by miRNAs through post-transcriptional mechanisms (see, Lewis et al. (2005) Cell 120: 15-20).
The number of targets is likely to increase when taking into account widespread unannotated transcription (see, Willingham and Gingeras (2006) Cell 125: 1215-1220) thus making these molecules a powerful regulatory class with the potential to intercept a wide network of fundamental cellular processes. Over the past several years an increasing number of miRNAs have been implicated in cancer development with mechanisms ranging from copy number alterations/mutations/epigenetic silencing or dysregulated transcriptional control of miRNA loci (see, Esquela-Kerscher and Slack (2006) Nat Rev Cancer 6: 259-269 and Garzon R, Cahn G A and Croce C M (2009) MicroRNAs in Cancer. Annu Rev Med 60: 167-179.)
These data reveal the oncogenic and tumor suppressive nature of miRNAs and highlight the correlation between various cancers and differential miRNA signatures. The ability to profile miRNAs in circulation thus represents a non-invasive opportunity to investigate disease specific miRNAs and is a promising alternative approach to current strategies for cancer surveillance.
A critical prerequisite for developing circulating miRNA-based diagnostics is the ability to accurately isolate and measure representative miRNA species in biofluids. In spite of high concentration of RNAses in plasma and serum, circulating miRNAs are surprisingly tractable. A key molecular property of these species is that they are highly stable in circulation and can survive unfavorable physiological conditions such as extreme variations in pH and multiple freeze thaw cycles (see Chen et al. (2008) Cell Res 18: 997-1006 and Mitchell et al. (2008) Proc Natl Acad Sci USA 105: 10513-10518).
Circulating miRNA may also be tumor derived, thus directly reflecting disease burden and are protected from degradation though inclusion in RNA binding proteins (see Wang et al. (2010) Nucleic Acids Res 38: 7248-7259) or sub-cellular particles (see Kosaka et al. (2010) Cancer Sci 101: 2087-2092 and Wang et al. (2010) Nucleic Acids Res 38: 7248-7259 and Valadi et al. (2007) Nat Cell Biol 9: 654-659) distinct from the hematopoietic cellular population.
All of these cellular attributes are susceptible to a variety of pre-analytic factors involving sample collection, processing, storage and extraction methods that can determine both the quantitative and qualitative effectiveness of this species for clinical use. In an effort to standardize results and bring uniformity to data quality several studies over the past few years have begun to explore and put forward recommendations for a subset of these pre-analytical variables (see Chen et al. (2008) Cell Res 18: 997-1006, Mitchell et al. (2008) Proc Natl Acad Sci USA 105: 10513-10518 and Chiu et al. (2001) Clin Chem 47: 1607-1613).